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Last Updated: August 14, 2020

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Claims for Patent: RE32271

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Summary for Patent: RE32271
Title: Isolation of plasminogen activators useful as therapeutic and diagnostic agents
Abstract:The existence of high fibrin-affinity urokinase is discovered by an isolation procedure using fibrin precipitated on an adsorptive-solid matrix. By the method described, the high affinity form of plasminogen activator can be isolated directly from urine or from kidney tissue culture medium. The method is economical and provides a relatively high yield of the activator. The high affinity that this plasminogen activator has for fibrin is a property that makes it an improved thrombolytic agent and when radiolabelled provides a new diagnostic agent for the specific detection of fibrin thrombi through nuclear scanning. The newly-isolated plasminogen activator has the following characteristics: a molecular weight of about 56,000 Daltons, a specific activity of about 40,000-50,000 CTA units/mg, the appearance of a single chain structure and a high affinity for fibrin.
Inventor(s): Husain; Syed S. (Newton, MA), Lipinski; Boguslaw (Newtonville, MA), Gurewich; Victor (Cambridge, MA)
Assignee:
Application Number:06/727,807
Patent Claims:1. The method of isolating a plasminogen activator from urine or culture medium, comprising

providing an adsorptive matrix having fibrin precipitated on its surface,

exposing a mother liquid based upon urine or culture medium and containing high fibrin-affinity plasminogen activator to the fibrin-containing matrix, whereby those plasminogen activator molecules which have high affinity therefor are bound to molecules of fibrin,

removing the remaining mother liquid, and

separating the plasminogen activator from the fibrin.

2. The method of claim 1 wherein the plasminogen activator is eluted from the fibrin surface by an eluant agent solution containing a member of the group consisting of arginine, lysine, and .epsilon.-amino-caproic acid.

3. The method of claim 1 wherein said matrix comprises diatomaceous earth.

4. The method of claim 1 wherein, for providing said matrix with fibrin on its surface, fibrinogen is treated with thrombin in the presence of the said matrix in a manner to cause fibrin to be precipitated upon said matrix without gel formation.

5. The method of claim 4 comprising exposing the adsorptive surface of said matrix to fibrinogen in quantity sufficient of effectively cover said adsorptive surface, thereafter introducing thrombin to convert said adsorbed fibrinogen to fibrin, whereby said adsorptive surface is effectively fully occupied by adsorbed fibrin.

6. The method of isolating urokinase comprising

providing a solid matrix with fibrin by treating fibrinogen with thrombin in the presence of said matrix to cause fibrin to be in an absorbed state upon said substrate,

exposing urine or culture medium to the substrate whereby a species of urokinase which has affinity towards fibrin is bound to the fibrin,

removing the unbound material by washing with a buffer solution,

separating the urokinase from the fibrin first by eluting the urokinase from the fibrin surface by an eluant agent comprising a member of the group consisting of arginine, lysine, .epsilon.-amino-caproic acid, and thereafter separating said urokinase from said agent.

7. The method of claim 1 or 6 wherein said matrix comprises particles wherein the liquid is exposed to said fibrin-carrying matrix by mixing said particles with said liquid.

8. The method of claim 1 or 6 wherein said liquid is removed by decanting and filtering followed by repeated washing with a buffer.

9. A plasminogen activator enzyme concentrate isolated from a biological source by the methods of any of the claims 1-6 and comprising urokinase (human) of molecular weight of about 56,000 Daltons, having high affinity for binding to fibrin on an adsorptive matrix and having the appearance of a single chain molecular structure.

10. .[.A.]. .Iadd.Urokinase .Iaddend.plasminogen activator isolated from .[.a biological source such as.]. urine or culture consisting essentially of urokinase (human) characterized as (a) having a molecular weight of about 56,000 Daltons .Iadd.as determined by gel-filtration.Iaddend., (b) .[.having a specific activity of less than 50,000 CTA units/mg when assayed on a fibrin plate, (c) having the appearance of a single chain structure corresponding to a molecular weight of 56,000 Daltons as evidenced by.]. .Iadd.appearing as .Iaddend.a single protein band .Iadd.corresponding to a molecular weight of about 56,000 Daltons .Iaddend.on 7.5 percent polyacrylamide gel when sodium dodecyl sulfate-gel electrophoresis is performed on unreduced urokinase, .[.d.]. .Iadd.(c) .Iaddend.retaining .[.the.]. said appearance .[.of a single chain structure as evidenced by a single protein band on 7.5 percent polyacrylamide gel when sodium dodecyl sulfate-gel.]. .Iadd.when said .Iaddend.electrophoresis is performed on urokinase which has been reduced by 0.1M dithiothreitol .Iadd.thereby indicating its single chain structure .Iaddend.and .[.(e).]. .Iadd.(d) .Iaddend.displaying a high binding affinity for fibrin precipitated on diatomaceous earth. .Iadd.11. The urokinase of claim 10 in lyophilized form..Iaddend.

Details for Patent RE32271

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Microbix Biosystems KINLYTIC urokinase INJECTABLE;INJECTION 021846 001 1978-01-16   Start Trial 2003-10-28 DISCN search
Microbix Biosystems KINLYTIC urokinase INJECTABLE;INJECTION 021846 002 1978-01-16   Start Trial 2003-10-28 DISCN search
Microbix Biosystems KINLYTIC urokinase INJECTABLE;INJECTION 021846 003 1978-01-16   Start Trial 2003-10-28 DISCN search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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